Instrumental method for plating and counting aerobic bacteria

ABSTRACT

A MACHINE FOR DEPOSITING A VARYING AMOUNT OF BACTERIA SOLUTION ON THE SURFACE OF A SOLIDIFIED AGAR PLATE IN THE CONFIGURATION OF AN ARCHIMEDES SPIRAL. THE AMOUNT OF SOLUTION DEPOSITED ON THE AGAR IS CONTINUOUSLY DECREASED, RESULTING IN A HIGHER CONCENTRATION OF BACTERIA PER UNIT LENGTH AT THE CENTER OF THE SPIRAL AND A DECREASING CONCENTRATION PER UNIT LENGTH AT THE EDGE OF THE PLATE. AFTER AN INCUBATION PERIOD THE BACTERIA BECOMES VISIBLE, AND ARE COUNTED BY INTERRUPTING A LIGHT BEAM INCIDENT TO A PHOTODIODE. THE CONCENTRATION OF AN UNKNOWN SOLUTION MAY BE DETERMINED BY VISUALLY COUNTING THE NUMBER OF COLONIES ON A DISCRETE AREA OF THE PLATE.

J. E. CAMPBELL ETAL 3,799,844

March 26, 1974 INST UME TA METHOD FOR PLATING AND COUNTING AEROBIC BACTERIA 2 Sheets-$heet 1 Filed June 2, 1971 INVENTORS UE/Es E.GILCHRIST a JEPTHA E.CAMPBELL March 26, 1974 J, E. CAMPBELL ETAL 3,799,844

INSTRUMENTAL METHOD FOR PLATTNG AND COUNTING AEROBIC BACTERIA Filed June 2, 1971 2 Sheets-Sheet 2 55: :::::E:. EN Q M W]. W

INVENTORS JAMES E. GILCHRIST 8x JEPTHA E. CAMPBELL BYM MWZ ATTORNEYS United States Patent 3,799,844 INSTRUMENTAL METHOD FOR PLATING AND COUNTING AEROBIC BACTERIA Jeptha E. Campbell and James E. Gilchrist, Cincinnati, Ohio, assignors to the United States of America as represented by the Secretary of the Department of Health, Education, and Welfare Filed June 2, 1971, Ser. No. 149,137 Int. Cl. C12k 1/00 US. Cl. 195127 5 Claims ABSTRACT OF THE DISCLOSURE A machine for depositing a varying amount of bacteria solution on the surface of a solidified agar plate in the configuration of an archimedes spiral. The amount of solution deposited on the agar is continuously decreased, resulting in a higher concentration of bacteria per unit length at the center of the spiral and a decreasing concentration per unit length at the edge of the plate. After an incubation period the bacteria becomes visible, and are counted by interrupting a light beam incident to a photodiode. The concentration of an unknown solution may be determined by visually counting the number of colonies on a discrete area of the plate.

BACKGROUND OF THE INVENTION The present invention relates to the quantifying of viable particles and, more particularly, to a device for depositing aerobic bacteria from a sample of unknown concentration on a solid culture medium in a variable amount for culturing followed by counting the colonies to determine the bacterial concentration of the original sample.

In the science of microbiology, Which by its very definition is that branch of knowledge which concerns living micro-organisms, there is a never-ending need to observe, study, catalog, and learn about bacteria, how they multiply, the size and operation of their colonies, their incubation periods, etc. There frequently arises, either in scientific microbiologic inquiries or in the field of medicine (e.g. for antibiotic sensitivity assay), the need to determine the total number of bacteria present in a sample, or the number of bacteria colonies which have incubated.

The normal procedure for determining the quantity of bacteria in an unknown solution, known as the pour plate method, is complex, involving making serial dilutions with multiple pipetting, and mixing each dilution with sterile liquid agar. A series of agar plates, each containing 'a different concentration of the unknown solution are prepared (under sterile conditions to avoid the introduction of outside bacteria), and these are then cultured. The plates that contain too many or too few bacterial colonies are discarded and the one containing a countable number is visually counted. The colony count obtained is multiplied by an appropropriate dilution factor to obtain the bacterial concentration in the sample.

The present invention overcomes many of these weaknesses and shortcomings in that it quickly and accurately deposits a small amount of the liquid of unknown concentration on the surface of an agar plate in a spiral of varying quantity. Pro-prepared agar plates, normally used only for qualifying procedures are thereby useful for quantifying determinations. Only one agar plate is needed, instead of many, and the preparation of the plate may be carried out in only about two minutes, some four to five times faster than the time necessary to prepare each separate plate by the conventional method using serial dilutions.

The present invention in overcoming many of the above weaknesses and shortcomings inherent in the prior 3,799,844 Patented Mar. 2 6, 1974 ice art equipment, quickly and accurately determines aerobic bacteria present in a sample with a minimum of human error. A varying amount of solution is deposited on the surface of a solidified agar plate in the con-figuration of an archimedes spiral. The amount of solution being deposited on the agar is continuously decreasing, resulting in a higher concentration of bacteria per unit length at the center of the spiral and a decreasing concentration per unit length as the edge of the plate is approached. The plates are then incubated for the bacterial colonies to become visible, the time necessary for this being dependent upon the bacterial species.

Since the amount of liquid deposited on the agar is known, the bacterial concentration may be calculated by counting the bacterial colonies along any line or group of lines. The unknown plate may also be compared to a standard set of plates by visually matching the whole plate or any comparable portion of the plates. The number of bacterial colonies may be electronically counted by shining a light through the agar plate and onto a photodiode, the change in transparency of the plate as caused by the bacteria registering on the photodiode to activate a counting circuit.

It is, accordingly, an object of the present invention to obviate the deficiencies of the prior art, as indicated above.

An object of the present invention is the provision of a method and apparatus for plating and counting aerobic bacteria.

Another object of the present invention is the provision of a device for automatically depositing solution in a varying amount on the surface of an agar plate.

Still another object of the invention is the provision of a means for depositing the solution on the agar plate in the configuration of an archimedes spiral.

Yet another object of the invention is the provision of a means for counting bacteria using a light and a sensor having a photodiode.

Still another object of the invention is the provision of means for counting bacteria wherein a sensor activates an electronic counter.

Another object of the invention is the provision of a means for counting aerobic bacteria which is fast, accurate, and minimizes human error in its operation.

Other objects and many of the attendant advantages of this invention will be readily appreciated as the same becomes better understood by reference to the following detailed description of a specific embodiment when considered in connection with the accompanying drawings in which like reference numerals designate like parts throughout the figures thereof and wherein;

FIG. 1 shows a perspective view of a device used for depositing solution on an agar plate;

FI 2 shows a front elevation of a device used for counting bacteria by reflected light; and

FIG. 3 is a partly schematic view of the invention showing another device for counting bacteria by transmitted light, and showing in greater detail the arrangement of the various components.

Referring now to the drawings there is shown a frame 10 which is substantially U-shaped and which rests on its horizontal part with its legs extending vertically. Joining the two vertical legs near their upper ends i at least one horizontal crossbar 11, the bar being held in place in the vertical legs by end caps or nuts 12. Bolted to the bottom of the frame 10 is an electric motor 13, the end of motor 13 having attached thereto a gearbox 14 which in used to rotate a disk 15. The disk 15 may be made of some light Weight metal such as aluminum and is so mounted on gearbox 14 that it is positioned horizontally and rotates about a vertical axis incorporated in the gearbox.

Bolted to the disk so as to be integral and rotate with it is a height adjusting means 16, this means having an adjusting knob 17 which turns a rack and pinion or equivalent arrangement (not shown) to move a plunger 18 up and down. Screwed to the end of the plunger 18 is a disk 20, preferably of transparent plastic also positioned in a horizontal plane, and movable vertically when the plunger 18 is moved. The transparent plastic disk 20 has positioning studs 21 around its periphery, of a depth to hold a Petri dish 22, so that the dish will be centered when disk 20 rotates, even though this rotation will not be at a high rate.

Suspended from the crossbars 11 is a moving platform 23 having an end bracket 24 at each of its ends for support. End brackets 24 have a transverse hole drilled therethrough of sufficient size to accommodate the crossbar 11, and linear ball bearings 25 within each bracket 24 provides a sliding fit so that moving platform 23 can freely move along the crossbars. Also forming a part of the left bracket 24 there is a fixed half-nut 26, this half-nut encircling and cooperating with a lead screw 27 in the usual manner to move the platform 23 when the lead screw is turned. The opposite end of lead screw 27 from half-nut 26 is rotatably mounted in the vertical leg of frame 10.

Forming a part of, and integrally mounted on, the moving platform 23 there is a rack and pinion 28 which may serve as a holder for the hereafter described syringe used in applying solution to Petri dish 22, as will become apparent hereinafter.

A bracket 30 is provided for furnishing support toa light source 31 and to an electronic sensor 32. In the preferred construction of FIG. 3 a portion of metal bracket 30 is C-shaped so that it extends on either side of the transparent disk 20, the light source 31 being on one end of the C, and below disk 20, while the other leg of the C holds the sensor 32 above disk 20. It is obvious through this arrangement that light from source 31 shines through disk 20 and onto the phototube 33, the amount of light striking the phototube being in direct proportion to the number of aerobic bacterial colonies which have formed on Petri dish 22. In some instances where counting by reflected light is satisfactory, however, the light source may be above the disk 20 as shown in FIG. 2 in which case it is not necessary to provide a transparent disk and dish.

Immediately below, and fastened to, disk 15 there is a pulley 34 and a beaded chain 35 or the equivalent, the chain 35 passing around an idler wheel 36 and ultimately around a smaller pulley 37 (FIG. 3) or directly to a larger pulley 41 (FIGS. 1 and 2). If desired, integral with pulley 37 there may be provided a slightly larger pulley 38 so that when these two pulleys turn a speed ratio output is obtained as shown in FIG. 3. In this embodiment, a second beaded chain 40 links a pulley 38 with the larger pulley 41, the latter pulley 41 being attached to lead screw 27 to serve as a means for rotating the lead screw. 7

Wires 42 carry a signal generated by a sensor 32 to a driving means 43, shown in block form, which may be of any familiar configuration, such as, for example, a Zener preamplifier, a Schmitt trigger, and a driver amplifier. The output of driving means 43 is connected to a coupler 44 and thence to an electronic counter 45, which may be any of the devices well known in the art. Should it be desirable, an oscillator 46 may be switched into the circuitry to enter a standard rate of counts (about 1,000 Hz./ second) into the electronic counter when the photodiode is conducting. Micro switches are preferably used to both stop the signal to the electronic counter and the motor when the sensor approaches the edge of the Petri dish; these are preferably provided on the horizontal cross-bars 11.

Referring now to FIG. 1 the invention is shown as it would b mod fied s igh y to a o pl h plating f a agar or other nutrient plate (Petri dish) with solution in a spiral configuration. The components bear the same part numbers a previously described with the exception that there is provided a coating device instead of sensing apparatus.

A syringe 49 is mounted on the platform 23, while a hollow syringe plunger 49' is contained therein for vertical movement, the plunger 49' being mounted on a mechanical device 48 including an arm 48', which device 48 is in turn carried on the mounting post 28 for vertical movement in a dovetail-like groove in the mounting post 28. A valve 47 is mounted at the top of the plunger 49' on the device 48. On the end of the arm 48 there is a pointer 50 which rides along the contoured surface of a cam 51, the purpose of which is to control the movement of the plunger 49" in the syringe 49. A plastic tube 52 joins a vacuum flask 53 and valve arrangement 47 for connecting the tube 52 to a source of vacuum through the vacuum flask 53.

A second tube 52' passes from the bottom of the syringe 49 and then through a rigid support tube 56 from which it extends terminating in a tip 54 which rides on the agar plate 22. The rigid support tube 56 is supported in turn by a substantially horizontal pivot 58 which is preferably parallel to the cross-bars 11. The weight of tube 56 maintains the tip 54 against the agar plate 22. A cut-off 55 is furnished to control the valve 47 and seal off any application of vacuum from passing through the 1 plunger 49' and the tube 52'.

The view given in FIG. 2 shows how agar plate 22 looks after the solution has been deposited on it. Also shown is another embodiment of electronic counter 45 with its driver 43.

Turning now to the operation of the device, and particularly that of depositing solution on an agar plate as shown in FIGS. 1 and 2, the electric motor 13, bolted to a U-shaped frame 10, operates the gear box 14 to turn a disk 15 at a predetermined rotary speed. Atop the height adjusting means 16 and turning with it is the transparent disk 20 which holds a Petri dish, or agar plate 22, the plate also being rotated by the motor. The moving platform 23' moves across the frame 10 along crossbars 11 under the influence of the lead screw 27 and half-nut 26, the platform being driven by motor 13, beaded chain 35, pulleys B7 and 38, beaded chain 40 and pulley 4'1 fastened to the lead screw. From this structure it is ob- 'vious that the platform moves over the agar plate at a speed which bears a definite and predetermined relation to the rate of rotation given the plate. Mounting post 28, secured to platform 23, has the extended arm 48' attached to it, so that as the platform moves, pointer 50 follows along the face of cam 51 and operates to depress the plunger 49 of the syringe 49 as the platform moves over the agar plate. The syringe 49 dispenses fluid at a rate determined by the configuration of the cam 51, to the surface of the agar plate through the tip 54 of plastic tube 52', positioned on the plate, tip 54 functioning as a moving stylus.

The plunger 49 of syringe 49 is hollow with valve 47 mounted on the upper end, so that when a vacuum is applied through the valve from tube 52, fluid may be introduced through tip 54 and tube 52 to back-flow into the syringe. A new sample is introduced through the plastic tube 52' in this manner with a minimum of contamination. When the syringe is filled, valve cut-off 55 is closed and the system is ready to dispense fluid on the agar plate.

Since the movement of platform 23 bears a fixed relationship to the rotation of the agar plate 22, it is clear that fluid dispensed from tip 54 onto the plate follows the configuration of an archimedes spiral, and since the plunger 49' of syringe 49 is being depressed by arm 48 the amount of solution being deposited on the agar is continuously decreasing, resulting in a higher concentrate tion of bacteria per unit length at the center of the spiral and a decreasing concentration per unit length as the edge of the plate is approached. After coating with bacterial solution, the plate must be incubated for the bacterial colonies to become visible, the time necessary for this being dependent upon the bacterial species.

Since the amount of liquid deposited on the agar is known, the bacterial concentration may be calculated by counting the bacterial colonies along any line or group of lines. The unknown plate may also be compared to a standard set of plates by visually matching the whole plate or any comparable portion of the plates. Bacterial sensitivity to antibiotics could be determined by applying bacterial solutions to a plate having various quantities of antibiotics.

The number of bacterial colonies may be counted electronically using a miniature photoelectric cell in conjunction with an electronic counter. This counting is accomplished by the arrangement shown in FIG. 3 wherein a sensor 32, having a photodiode 33 in its tip, is mounted on platform 23 so that it moves across the agar plate, light from source 31 shining through transparent disk 20 and the agar plate to impinge on the sensor. The movement of the sensor over a bacterial colony causes a change in the intensity of the light on the photodiode. This causes the photodiode to conduct and send a signal through driving means 42, coupler 44, and on to electronic counter 45.

Two modes of counting may be recorded on counter 45: (1) the total number of individual signals generated by the sensor, and (2) the total length of time the sensor is conducting.

From the above description of the structure and oper ation of the invention it is clear that the device ofiers many improvements over the shortcomings and Weaknesses of the prior art apparatus. It is obvious that the invention provides a method and apparatus for quickly, easily, and accurately determining bacterial colonies with a minimum of contamination, the result being a finite number rather than one obtained by estimation and human error. The device is successfully used over a bacterial concentration range of at least 100 to 2. bacteria per ml.

Obviously many modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the invention may be included modifications and variations other than as specifically described. For example, means other than the lead screw 27 may be used to advance the platform 23, such as a gear and movable rack.

What is claimed is:

1. Apparatus for plating aerobic bacteria in a constantly varying concentration on a growth medium to determine bacterial concentration, comprising:

mounting means for mounting a plate for rotation,

said plate containing a bacterial growth medium; rotating means connected to said mounting means for rotating said growth plate;

a bacterial solution dispensing means above said bacterial growth plate for dispensing bacterial solution onto the growth medium, said dispensing means having a dispensing tube terminating in a dispensing p;

means for pivotably supporting said dispensing tube at the dispensing tip end thereof such that said tip continuously rides on the medium when in use;

means for moving said dispensing means diametrically across said bacterial growth plate as the plate rotates so that the solution is deposited on the plate in a spiral configuration; and

means for mechanically varying, continuously, the amount of the deposited solution as said dispensing means moves across said bacterial growth plate.

2. The apparatus of claim 1 wherein said mounting means and rotating means for the bacterial growth plate comprises a motor;

a gearbox attached to the motor;

a circular disk driven by the gearbox;

height adjusting means mounted on the circular disk;

and

a transparent plastic disk mounted on the height adjusting means.

3. The apparatus of claim 1 wherein said dispensing means further comprises a syringe having a hollow movable plunger and means to cause fluid to backflow into the syringe and through the plunger;

wherein said dispensing tube is connected to said syringe at the end thereof opposite the dispensing tip.

4. The apparatus of claim 2 wherein said means for moving the dispenser across the bacterial growth plate comprises a moving platform;

means mounted on the platform for supporting the dispenser;

a worm drive connected to the platform to move the platform transversely across the bacterial growth plate; and

tflexi-ble means connecting the worm drive and the motor gearbox to turn the worm drive.

5. The apparatus of claim 3 wherein said means for varying the amount of the deposited solution comprises a pivoted arm connected to the plunger of the syringe;

a cam plate having a sloping face; and

a pointer on the free end of the pivoted arm to follow the cam plate whereby the plunger is decreasingly depressed by the pivoted arm as the platform moves and the pointer travels down the sloping face of the cam plate.

References Cited R. E. Trotman, Automation, Mechanization and Data Handling in Microbiology, Academic Press, pp. 211-221; 1970.

L. S. Gall et al., Developments In Industrial Microbiology, Garamond/Pridemark Press, pp. 460-469; 1970.

LIONEL M. SHAPIRO, Primary Examiner R. J. WARDEN, Assistant Examiner 

